Synthesis and characterization of the sweet protein brazzein

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose, and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless. © 1996 John Wiley & Sons, Inc.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.     

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.     

Dietary polyunsaturated fatty acids suppress acute hepatitis, alter gene expression and prolong survival of female Long-Evans Cinnamon rats, a model of Wilson disease

In the Long-Evans Cinnamon rat, copper accumulates in the liver because of a mutation in the copper-transporting ATPase gene, and peroxidative stresses are supposed to be augmented. We examined the effects of dietary fatty acids on hepatitis, hepatic gene expression, and survival. Rats were fed a conventional, low-fat diet (CE2), a CE2 diet supplemented with 10 wt% of lard (Lar), high-linoleic soybean oil (Soy), or a mixture of docosahexaenoic acid (DHA)-rich fish oil and soybean oil (DHA/Soy). Among female rats, the mean survival times of the DHA/Soy and the Soy groups were longer by 17 approximately 20% than in the Lar and the CE2 groups. Among male rats, the survival times were much longer than in the females, but no significant difference in survival was observed among the dietary groups. Serum ceruloplasmin levels in female and male rats of all of the dietary groups were similar. Serum transaminase levels of the DHA/Soy group tended to be lower than in the CE2 group. Histological examinations revealed a marked degeneration in hepatic tissue integrity in the Lar and CE2 groups but not in the DHA/Soy group. Hepatic levels of metal-related genes, transferrin and ceruloplasmin, as well as those related to bile acid synthesis were up-regulated, and an inflammation-related gene (cyclooxygenase [COX]-2) was down-regulated in the DHA/Soy group. Some proliferation-related genes were also affected by the dietary fatty acids. These results indicate that polyunsaturated fatty acids suppress the development of acute hepatitis and prolong survival in females, regardless of whether they are of the n-6 or n-3 type, which are associated with altered gene expressions.     

Structure and direct electrochemistry of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7

Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity. P450st was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=53.6 A, b=55.1 A, and c=130.9 A, and the structure was determined at a 3.0 A resolution. The final R-factor was 0.194 (Rfree=0.235). Structural comparison with cytochrome P450 from S. solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl- binding site is responsible for the affinity for a ligand coordinating heme iron. Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated. A quasi-reversible redox response has been observed even at elevated temperatures of up to 80 degrees C.     

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.     

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.     

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.     

ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.